Volume 17, Issue 6, Pages 793-803 (March 2005) HIF-1α Induces Genetic Instability by Transcriptionally Downregulating MutSα Expression  Minori Koshiji, Kenneth K.-W. To, Stefanie Hammer, Kensuke Kumamoto, Adrian L. Harris, Paul Modrich, L. Eric Huang  Molecular Cell  Volume 17, Issue 6, Pages 793-803 (March 2005) DOI: 10.1016/j.molcel.2005.02.015 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Hypoxia Induces Genetic Instability by Specifically Inhibiting MutSα Expression in a p53-Dependent Manner (A) HCT116 cells were either subjected to hypoxia or infected with recombinant adenoviruses expressing GFP or ΔODD for 48 hr. The dinucleotide-repeat marker D17S250 was used for MIN analysis. Arrows denote induced MIN. (B) HEC59 cells were treated and analyzed as above. (C) In-frame (IF) and out-of-frame (OF) β-galactosidase reporters were used to examine hypoxia-induced genetic instability in cell types as indicated. The results were presented as means + standard deviations from three independent experiments in duplicate. *p < 0.05; **p < 0.01; RBU, relative β-galactosidase activity; N, normoxia; and H, hypoxia. (D) HCT116 cells were subjected to hypoxia for 16 hr and analyzed for hypoxic repression of MSH2 and MSH6 protein levels by Western blot analysis. HIF-1α, p53, and β-actin were included as controls. (E) Normal human small airway epithelial cells (SAEC) and fibroblasts (MRC5) were subjected to hypoxia as above. The mRNA levels of mismatch repair genes, as indicated, were determined with real-time PCR. PGK1 served as a positive control for hypoxic induction. Representative results from three independent experiments in triplicate were presented as means ± standard errors. (F) HCT116 (TP53+/+), its TP53−/− mutant, and HeLa cells were treated as above and examined with real-time PCR. Molecular Cell 2005 17, 793-803DOI: (10.1016/j.molcel.2005.02.015) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 HIF-1α Is Necessary and Sufficient to Induce Genetic Instability by Downregulating MutSα Expression (A) HCT116 cells were transfected with siRNAs targeting HIF1A or HIF2A and then subjected to hypoxic treatment. Luciferase siRNA (luc) was used as a negative control. MSH2, MSH6, and PGK1 mRNA levels were determined with real-time PCR. Representative results are shown as in Figure 1E. (B) Cells were treated as in (A) and assayed for specific protein levels, as indicated, by sequential probing of the same blot with the corresponding antibodies. (C) HCT116 cells were infected with adenoviruses expressing ΔODD (Ad-ΔODD) or transcriptionally inactive ΔODD (Ad-LCLL) for 16 hr. Cells without treatment (−) or infected with Ad-GFP served as controls. Transcripts of MSH2 and MSH6, along with HIF1A, ΔODD, hypoxia-inducible BHLHB2, and ACTB were determined with conventional PCR and quantified with densitometry. The numerical results in reference to ACTB levels are indicated. (D) HCT116 cells were subjected to the same treatment as above, and protein levels of specified genes were sequentially probed with specific antibodies. Molecular weights are indicated on the left. (E) HCT116+Ch3 cells were analyzed for MMR gene expression with real-time PCR after adenoviral infection as indicated. (F) Effects of Ad-ΔODD on genetic instability were examined with IF and OF reporters in HCT116 and HEC59 cells. The results were presented as in Figure 1C. **p < 0.01. Molecular Cell 2005 17, 793-803DOI: (10.1016/j.molcel.2005.02.015) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Requirement of Myc and p53 for MutSα Expression (A) HCT116 cells were transfected with MYC-specific siRNA and subsequently assayed for MutSα expression in normoxia and hypoxia with real-time PCR. Representative results are shown as in Figure 1E. (B) Effects of MYC siRNA were examined by sequential probing with respective antibodies in Western blotting. (C) TP53 expression was knocked down in HCT116 TP53+/+ cells by specific siRNA or restored in HCT116 TP53−/− cells with adenoviral infection. The mRNA levels of specified genes were determined by real-time PCR. (D) MSH2 and MSH6 protein levels were also examined in the cells treated as above by Western blot analysis. p53 levels were also determined. Molecular Cell 2005 17, 793-803DOI: (10.1016/j.molcel.2005.02.015) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Myc Displacement by Hypoxia from MSH2 and MSH6 Promoters Is p53 Dependent (A) Schematic representation of the MSH2 and MSH6 proximal promoters. Double-arrowed lines indicate regions that were PCR-amplified in ChIP. Representative data of the shadowed regions are shown in (B). (B) Chromatin immunoprecipitations of normoxic and hypoxic HCT116 cells were performed by using antibodies against indicated transcription factors. Both TP53+/+ and TP53−/− cells were used. Specific primers flanking regions of the MSH2, MSH6, and CDKN1A proximal promoters were used to amplify the immunoprecipitated genomic DNA. Sheared genomic DNA prior to immunoprecipitations served as input, and immunoglobulin (IgG) served as a control. Molecular Cell 2005 17, 793-803DOI: (10.1016/j.molcel.2005.02.015) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Sp1 Is Essential for Recruiting HIF-1α onto the MSH2 Promoter (A) HCT116 cells were transfected with SP1 siRNA, and subsequently the expression of MutSα genes was determined with real-time PCR. Representative results are shown as in Figure 1E. (B) Normoxic and hypoxic HCT116 cell lysates were immunoprecipitated with anti-Sp1 antibody (α-Sp1IP), followed by Western blotting with anti-Myc and anti-HIF-1α antibodies (bottom). Input levels of HIF-1α and Myc were determined by direct Western blot (top). HIF1A expression was also silenced by specific siRNA. (C) In vitro-translated, [35S]methionine-labeled (with asterisks) Myc or HIF-1α was coimmunoprecipitated with anti-Sp1 antibody against endogenous Sp1. The addition of unlabeled HIF-1α abrogated Myc coprecipitation, whereas the addition of unlabeled Myc had no effect on HIF-1α coprecipitation. Input: 10% of lysates without immunoprecipitations. (D) An MSH2 luciferase reporter was cotransfected into HCT116 cells with vectors expressing p53, Myc, or ΔODD. Cotransfection with pcDNA3 (−) was used as a control. The reporter activity was presented as relative luciferase activity (RLU) as means + standard deviations. An MSH2 mutant reporter (MSH2-Sp1) with a mutation at a putative Sp1 site was also examined. The data from three independent experiments in duplicate were presented as means + standard deviations. **p < 0.01. (E) MRC5 cell extract was assayed for Sp1 DNA binding with [32P]-labeled oligonucleotides containing a consensus Sp1 element (Sp1*), a putative Sp1 site in the MSH2 promoter (MSH2*), or a mutated Sp1 site (MSH2m*). A 100-fold excess of unlabeled oligonucleotides of the consensus Sp1 (Sp1), wild-type MSH2 (MSH2), or mutated MSH2 (MSH2m) was used for competition. An arrowhead denotes Sp1 binding and an arrow denotes Sp3 binding. (F) Normoxic and hypoxic MRC5 extracts were subjected to supershift with the addition of antibodies against Sp1 (α-Sp1), HIF-1α (α-HIF), and Sp3 (α-Sp3). Minus symbol, no antibody added. Molecular Cell 2005 17, 793-803DOI: (10.1016/j.molcel.2005.02.015) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 The N-Terminal HIF-1α Interacts with Sp1 and Induces Genetic Instability (A) HIF-1α 1–329, HIF-1α 1–167, and ΔODD and its R27G mutant were translated in vitro and subjected to anti-Sp1 immunoprecipitation. Input: 10% of lysates without immunoprecipitations. (B) HIF-1α 1–329 and the above mutants were tested for their effect on wild-type MSH2 promoter (MSH2) and the promoter with a mutation at the Sp1 binding site (MSH2-Sp1). The results were presented as in Figure 5D. **p < 0.01. (C) HIF-1α 1–329 and its R27G mutant were examined together with other HIF-1α mutants as specified for their ability to induce genetic instability with the IF and OF reporters. The results were presented as in Figure 1C. Molecular Cell 2005 17, 793-803DOI: (10.1016/j.molcel.2005.02.015) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 Elevated HIF-1α Levels in Human Sporadic Colon Cancers Are Inversely Correlated with MSH2 Expression (A) Immunohistochemical staining of the colon cancer tissues with antibodies against p53 (a and d, undetectable; g and j, detectable), HIF-1α (b and h, negative; e and k, positive), and MSH2 (f, negative; c, i, and l, positive). The ID numbers corresponding to the specimens were listed on top. (B) A total of 80 specimens was tabulated according to the immunohistochemical staining of HIF-1α and MSH2 (top), or stratified based on the immunohistochemical detection of p53 (middle and bottom) and tabulated as above. p53 undetectable denotes p53 wild-type or loss of expression, and p53 detectable indicates p53 mutations. The data were analyzed with Fisher’s exact test for two-tailed p values. The association between HIF-1α and MSH2 was indicated by an odds ratio (OR). Molecular Cell 2005 17, 793-803DOI: (10.1016/j.molcel.2005.02.015) Copyright © 2005 Elsevier Inc. Terms and Conditions